The structural basis of the difference in sensitivity for PNGase F in the De-N-glycosylation of the native bovine pancreatic ribonucleases B and BS

Abstract

In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH−CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21–124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase Bcm). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase Bcm. NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the α-helix of the amino acid residues 1–20 has a stabilizing effect. In RNase BS, the α-helix formed by amino acid residues 23–32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS−PNGase F complex is proposed.